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Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

Guan Y, Meurer M, Raghavan S, Rebane A, Lindquist JR, Santos S, Kats I, Davidson MW, Mazitschek R, Hughes TE, Drobizhev M, Knop M, Shah JV - Mol. Biol. Cell (2015)

Bottom Line: We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5.We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair.The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.

No MeSH data available.


Related in: MedlinePlus

Intracellular induced dimerization measured by FCCS. (A) Schematic of induced heterodimerization of hmKeima8.5-FKBP12 and mTFP1-FRB by rapamycin. (B) Cross-correlation curves, comparison before and after addition of rapamycin. (C) Rapamycin induced dimerization measured by the percentage of dual-color molecules over total molecule number of hmKeima8.5. The binding of FKBP12 and FRB causes increase in dual-color percentage. A control construct (mTFP1-P2A-hmKeima8.5) under rapamycin addition is unchanged. (D) Time course of heterodimerization in individual cells upon addition of different concentrations of rapamycin. (E) Two cross-correlation curves from cell 1 at 45 and 75 min after treatment with 100 nM rapamycin. (F) Schematic of association of Coumarin 343–conjugated monofunctional binder (SAS121) with FKBP12-hmKeima8.5, followed by competition with excess nonfluorescent monofunctional binder (MAZ1258). (G) FCCS Measurements show the complex of SAS121 with FKBP12-hmKeima8.5 and dissociation upon excess addition of MAZ1258.
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Figure 6: Intracellular induced dimerization measured by FCCS. (A) Schematic of induced heterodimerization of hmKeima8.5-FKBP12 and mTFP1-FRB by rapamycin. (B) Cross-correlation curves, comparison before and after addition of rapamycin. (C) Rapamycin induced dimerization measured by the percentage of dual-color molecules over total molecule number of hmKeima8.5. The binding of FKBP12 and FRB causes increase in dual-color percentage. A control construct (mTFP1-P2A-hmKeima8.5) under rapamycin addition is unchanged. (D) Time course of heterodimerization in individual cells upon addition of different concentrations of rapamycin. (E) Two cross-correlation curves from cell 1 at 45 and 75 min after treatment with 100 nM rapamycin. (F) Schematic of association of Coumarin 343–conjugated monofunctional binder (SAS121) with FKBP12-hmKeima8.5, followed by competition with excess nonfluorescent monofunctional binder (MAZ1258). (G) FCCS Measurements show the complex of SAS121 with FKBP12-hmKeima8.5 and dissociation upon excess addition of MAZ1258.

Mentions: A practical intracellular application of single-excitation 2PE-FCCS was tested in cells by expressing the hmKeima8.5-(GGGS)2-FKBP12 and mTFP1-(GGGS)2-FRB with P2A linker. The schematic in Figure 6A shows how rapamycin can induce the formation of a dual-color complex between hmKeima8.5-FKBP12 and TFP-FRB. On the addition of 1 μM rapamycin, the cross-correlation signals increased over pretreatment signals when measured in the same cell (Figure 6B). There were no cross signals in the control construct of mTFP1-P2A-hmKeima8.5 before or after rapamycin treatment (Figure 6C).


Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

Guan Y, Meurer M, Raghavan S, Rebane A, Lindquist JR, Santos S, Kats I, Davidson MW, Mazitschek R, Hughes TE, Drobizhev M, Knop M, Shah JV - Mol. Biol. Cell (2015)

Intracellular induced dimerization measured by FCCS. (A) Schematic of induced heterodimerization of hmKeima8.5-FKBP12 and mTFP1-FRB by rapamycin. (B) Cross-correlation curves, comparison before and after addition of rapamycin. (C) Rapamycin induced dimerization measured by the percentage of dual-color molecules over total molecule number of hmKeima8.5. The binding of FKBP12 and FRB causes increase in dual-color percentage. A control construct (mTFP1-P2A-hmKeima8.5) under rapamycin addition is unchanged. (D) Time course of heterodimerization in individual cells upon addition of different concentrations of rapamycin. (E) Two cross-correlation curves from cell 1 at 45 and 75 min after treatment with 100 nM rapamycin. (F) Schematic of association of Coumarin 343–conjugated monofunctional binder (SAS121) with FKBP12-hmKeima8.5, followed by competition with excess nonfluorescent monofunctional binder (MAZ1258). (G) FCCS Measurements show the complex of SAS121 with FKBP12-hmKeima8.5 and dissociation upon excess addition of MAZ1258.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Intracellular induced dimerization measured by FCCS. (A) Schematic of induced heterodimerization of hmKeima8.5-FKBP12 and mTFP1-FRB by rapamycin. (B) Cross-correlation curves, comparison before and after addition of rapamycin. (C) Rapamycin induced dimerization measured by the percentage of dual-color molecules over total molecule number of hmKeima8.5. The binding of FKBP12 and FRB causes increase in dual-color percentage. A control construct (mTFP1-P2A-hmKeima8.5) under rapamycin addition is unchanged. (D) Time course of heterodimerization in individual cells upon addition of different concentrations of rapamycin. (E) Two cross-correlation curves from cell 1 at 45 and 75 min after treatment with 100 nM rapamycin. (F) Schematic of association of Coumarin 343–conjugated monofunctional binder (SAS121) with FKBP12-hmKeima8.5, followed by competition with excess nonfluorescent monofunctional binder (MAZ1258). (G) FCCS Measurements show the complex of SAS121 with FKBP12-hmKeima8.5 and dissociation upon excess addition of MAZ1258.
Mentions: A practical intracellular application of single-excitation 2PE-FCCS was tested in cells by expressing the hmKeima8.5-(GGGS)2-FKBP12 and mTFP1-(GGGS)2-FRB with P2A linker. The schematic in Figure 6A shows how rapamycin can induce the formation of a dual-color complex between hmKeima8.5-FKBP12 and TFP-FRB. On the addition of 1 μM rapamycin, the cross-correlation signals increased over pretreatment signals when measured in the same cell (Figure 6B). There were no cross signals in the control construct of mTFP1-P2A-hmKeima8.5 before or after rapamycin treatment (Figure 6C).

Bottom Line: We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5.We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair.The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.

No MeSH data available.


Related in: MedlinePlus